Recent advances in the understanding of the molecular bases of disease states and conditions have permitted the rationally-based development, at least in principle, of therapies which are specifically designed to target a particular molecular entity or entities. Unfortunately, a practical difficulty often arises in attempting to treat diseases with rationally-designed drugs, viz., while the drug may work as expected in vitro, in order to have the desired therapeutic effect the drug must be able to reach the site of action in vivo without being metabolically inactivated or degraded. In the case of drugs which must reach an intracellular site to be effective, providing the drug in a form capable of reaching the desired site can be difficult. Although many proposals have been made to deal with this problem, there are few approaches which are broadly applicable to a wide variety of drugs and disease states. One approach has been to administer the drug in a form, such as a liposome preparation, which allows the drug to cross the cell membrane. However, non-targeted liposomes may deliver the drug to non-target cells or organs, and the use of specifically-targeted liposomes can be expensive or inconvenient.
The endocytotic pathway of many protein toxins comprising separate A (enzymatic) and B (receptor binding) subunits, involves cell binding, internalization, translocation from an intracellular compartment to the cytosol, and enzymatic inactivation of their intracellular targets (1, 2). After translocation to the cytoplasm, the A subunits of ricin, abrin, modeccin, and verotoxins catalytically inactivate the 28 S RNA of 60 S ribosomal subunits, leading to an inhibition of cellular protein synthesis (3, 4). In addition, both the holotoxin and the B subunit are capable of inducing programmed cell death (apoptosis)(5-7).
The E coli derived family of verotoxins (or Shiga-like toxins) comprise VT1, VT2 and VT2c, which are involved in the etiology of microvascular disease in man (8), primarily in the very young and elderly (9), and VT2e which causes edema disease in pigs (10). The glycolipid globotriaosylceramide (galal-4galbl-4glc cer.-Gb3 ) at the plasma membrane is the specific receptor for all verotoxins and mediates the internalization of verotoxin (VT1) into susceptible cells by capping and receptormediated endocytosis (RME) (11). Verotoxin is the only glycolipid binding ligand that is internalized into eukaryotic cells by means of RME (12-14). In addition to receptor concentration, both heterogeneous fatty acid composition of Gb3 (15, 16) and phospholipid chain length within the phospholipid bilayer (17) play important roles in binding and internalization of VT. Molecular modeling studies of the Gb3 binding site in the B subunit (18) show that different conformers of membrane Gb3 may bind in different sites. Such conformers may be related to the Gb3 fatty acid content and membrane phospholipid microenvironment (18-20).
The requirement for retrograde transport for intoxication of cells by verotoxin was first demonstrated by Sandvig (21). A431 cells are resistant to VT. These cells expressed Gb3 but the toxin receptor-complex was internalized to endosomes and lysosomes. However, following growth in the presence of butyric acid, an inducer of cell differentiation, A431 cells became VT-sensitive, coincident with the detection of internalized toxin in Golgi cisternae, ER and even in the nuclear envelope (21). Similar targeting of both the holotoxin and B subunit to the nuclear envelope in highly toxin sensitive B lymphomas has been found (11).
In studying the sensitivity of human astrocytoma cell lines to verotoxin, significant differences which do not correlate with the level of receptor expression (6). Similarly, multiple drug resistant (MDR) variants of ovarian tumor cells lines were hypersensitive to VT as compared to the parental cell line, without major increase in receptor expression (22). Based on these discrepancies, Gb3-dependent intracellular traffic plays a major role in determining cell sensitivity to VT.
The invention relates to hybrid compounds, and methods of preparing and using the same.
In one aspect, the invention provides a hybrid molecule comprising a first domain and a second domain covalently linked, wherein (a) said first domain comprises a domain which is capable of specific binding to Gb3; (b) said second domain comprising a moiety selected from the group consisting of drug moiety, a nucleic acid, a probe, a polypeptide, and a hook, with the proviso that the second domain is not a verotoxin or a fragment thereof. In preferred embodiments, the first domain is a verotoxin or a verotoxin subunit; the first domain is VT-B; the second domain is a polypeptide; the polypeptide is a DNA binding element; the second domain is a nucleic acid; the nucleic acid is an antisense nucleic acid. In another aspect, the invention provides a pharmaceutical composition comprising a hybrid molecule of the invention and a pharmaceutically acceptable carrier.
In another aspect, the invention provides a method for modulating a cell-associated activity comprising contacting a cell with the hybrid molecule of the invention such that a cell associated activity is altered relative to the cell-associated activity of the cell in the absence of the hybrid molecule.
In another aspect, the invention relates to a method for directing the delivery of the hybrid compound of the invention to a particular intracellular location in a cell, the method comprising contacting the cell with the hybrid compound, optionally in the presence of a compound which alters fatty acid composition of Gb3, such that the hybrid compound is delivered to a particular intracellular location in the cell.
In another aspect, the invention provides a se of a hybrid compound of the invention for the manufacture of a medicament for treatment, prophylaxis, or diagnosis.